Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1,-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury , including diminished proteolysis of-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered also occurred in two models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

To defend against microbial invaders but also to establish symbiotic programs, plants need to detect the presence of microbes through the perception of molecular signatures characteristic of a whole class of microbes. Among these molecular signatures, extracellular glycans represent a structurally complex and diverse group of biomolecules that has a pivotal role in the molecular dialogue between plants and microbes. Secreted glycans and glycoconjugates like symbiotic lipochitooligosaccharides or immunosuppressive cyclic β-glucans act as microbial messengers that prepare the ground for host colonization. On the other hand, microbial cell-surface glycans are important indicators of microbial presence. They are conserved structures normally exposed and thus accessible for plant hydrolytic enzymes and cell-surface receptor proteins. While the immunogenic potential of bacterial cell-surface glycoconjugates such as lipopolysaccharides and peptidoglycan has been intensively studied in the past years, perception of cell-surface glycans from filamentous microbes such as fungi or oomycetes is still largely unexplored. To date, only few studies have focused on the role of fungal-derived cell-surface glycans other than chitin, highlighting a knowledge gap that needs to be addressed. The objective of this review is to give an overview on the biological functions and perception of microbial extracellular glycans, primarily focusing on their recognition and their contribution to plant-microbe interactions.

Soil-dwelling microbes are the principal inoculum for the root microbiota, but our understanding of microbe–microbe interactions in microbiota establishment remains fragmentary. We tested 39,204 binary interbacterial interactions for inhibitory activities in vitro, allowing us to identify taxonomic signatures in bacterial inhibition profiles. Using genetic and metabolomic approaches, we identified the antimicrobial 2,4-diacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites whose combined functions explain most of the inhibitory activity of the strongly antagonistic Pseudomonas brassicacearum R401. Microbiota reconstitution with a core of Arabidopsis thaliana root commensals in the presence of wild-type or mutant strains revealed a root niche-specific cofunction of these exometabolites as root competence determinants and drivers of predictable changes in the root-associated community. In natural environments, both the corresponding biosynthetic operons are enriched in roots, a pattern likely linked to their role as iron sinks, indicating that these cofunctioning exometabolites are adaptive traits contributing to pseudomonad pervasiveness throughout the root microbiota.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall (CW) layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counterdefensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

The primary visual cortex (area V1) is an extensively studied part of the cerebral cortex with well-characterized connectivity, cellular and molecular architecture and functions (for recent reviews see Amunts and Zilles, Neuron 88:1086–1107, 2015; Casagrande and Xu, Parallel visual pathways: a comparative perspective. The visual neurosciences, MIT Press, Cambridge, pp 494–506, 2004). In humans, V1 is defined by heavily myelinated fibers arriving from the radiatio optica that form the Gennari stripe in cortical layer IV, which is further subdivided into laminae IVa, IVb, IVcα and IVcβ. Due to this unique laminar pattern, V1 represents an excellent region to test whether multimodal mass spectrometric imaging could reveal novel biomolecular markers for a functionally relevant parcellation of the human cerebral cortex. Here we analyzed histological sections of three post-mortem brains with matrix-assisted laser desorption/ionization mass spectrometry imaging and laser ablation inductively coupled plasma mass spectrometry imaging to investigate the distribution of lipids, proteins and metals in human V1. We identified 71 peptides of 13 different proteins by in situ tandem mass spectrometry, of which 5 proteins show a differential laminar distribution pattern revealing the border between V1 and V2. High-accuracy mass measurements identified 123 lipid species, including glycerolipids, glycerophospholipids and sphingolipids, of which at least 20 showed differential distribution within V1 and V2. Specific lipids labeled not only myelinated layer IVb, but also IVa and especially IVc in a layer-specific manner, but also and clearly separated V1 from V2. Elemental imaging further showed a specific accumulation of copper in layer IV. In conclusion, multimodal mass spectrometry imaging identified novel biomolecular and elemental markers with specific laminar and inter-areal differences. We conclude that mass spectrometry imaging provides a promising new approach toward multimodal, molecule-based cortical parcellation.Electronic supplementary materialThe online version of this article (10.1007/s00429-018-1660-y) contains supplementary material, which is available to authorized users.

Carbon concentrating mechanisms enhance the carboxylase efficiency of the central photosynthetic enzyme rubisco by providing supra-atmospheric concentrations of CO2 in its surrounding. In the C4 photosynthesis pathway, this feat is realised by combinatory changes to leaf biochemistry and anatomy. In contrast to the C4 pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 to 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed ‘C3–C4 intermediates’. In the present study, we perform a physiological, biochemical and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation between tested plant species. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4 classified taxa indicating a crucial role of anatomical features for CO2 concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of PEPC activities and metabolite composition suggests that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct and fit photosynthesis type.

Carbon concentrating mechanisms enhance the carboxylase efficiency of the central photosynthetic enzyme rubisco by providing supra-atmospheric concentrations of CO2 in its surrounding. In the C4 photosynthesis pathway, this is achieved by combinatory changes to leaf biochemistry and anatomy. Carbon concentration by the photorespiratory glycine shuttle requires fewer and less complex modifications. It could represent an early step during evolution from C3 to C4 photosynthesis and an inspiration for engineering approaches. Plants displaying CO2 compensation points between 10 to 40 ppm are therefore often termed 'C3–C4 intermediates'. In the present study, we perform a physiological, biochemical and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathways showed considerable variation between the species but also within species. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4 classified accessions indicating a crucial role of anatomical features for CO2 concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual plant accessions, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of PEPC activities suggests that C4-like shuttles have not evolve in the investigated Brassicaceae.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner, insoluble cell wall (CW) layer and an outer, soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, implicating different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Additionally, exogenous application of β-GD leads to enhanced fungal colonization in barley. Our data highlights the hitherto undescribed capacity of this often overseen fungal EPS layer to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.